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T R Arnett

Publications and source records attributed to T R Arnett.

10 recordsLinked to original sources
PubMed1992

Interaction of androgen and 1,25-dihydroxyvitamin D3: effects on normal rat bone cells.

We studied the actions of testosterone (T) and 5 alpha-dihydrotestosterone (DHT) in combination with 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on primary rat bone cells. The actions of androgens were generally anabolic, although response patterns varied considerably between cultures. For example, DHT caused striking dose- and time-dependent increases in [3H]thymidine incorporation into calvarial cells over the range 1-100 nM, with maximal stimulation of 2.5-fold after 9 days in culture. Testosterone (50 nM) also stimulated [3H]thymidine incorporation into long bone-derived cells. 1,25-(OH)2D3 generally blunted or abolished the proliferative action of androgens but was not itself always inhibitory; in some experiments, stimulation of [3H]thymidine incorporation occurred. Collagen production, as assessed by [3H]proline incorporation into pepsin-resistant protein secreted by calvarial cells, was also stimulated by DHT. In some cultures, androgen responses were absent, although striking inhibitory responses to 1,25-(OH)2D3 were observed. These results illustrate complex patterns of responses to androgens and 1,25-(OH)2D3 in cells derived from rat bone.

Alkaline Phosphatase↗
PubMed1992

Rapid neural growth: calcitonin gene-related peptide and substance P-containing nerves attain exceptional growth rates in regenerating deer antler.

Deer antler is a unique mineralized tissue which can produce very high growth rates of > 1 cm/day in large species. On completion of antler growth, the dermal tissues which cover the antler are shed and the underlying calcified tissue dies. After several months the old antler is discarded and growth of a new one begins. It is known that deer antlers are sensitive to touch and are innervated. The major aims of this study were to identify and localize by immunohistochemical techniques the type of innervation present, and to find out whether nerve fibres could exhibit growth rates comparable to those of antler. We have taken tissue sections from the tip and shaft of growing Red deer (Cervus elaphus) antlers at three stages of development; shortly after the initiation of regrowth, the rapid growth phase, and near the end of growth. Incubation of tissue sections with antisera to protein gene product 9.5 (a neural cytoplasmic protein), neurofilament triplet proteins (a neural cytoskeletal protein), substance P and calcitonin gene-related peptide (both of which are present in and synthesized by sensory neurons) showed the presence of immunoreactive nerve fibres in dermal, deep connective and perichondrial/periosteal tissues at all stages of antler growth. The sparse distribution of vasoactive intestinal polypeptide-like immunoreactivity was found in dermal tissue only at the earliest stage of antler development. Nerve fibres immunoreactive to neuropeptide Y, C-flanking peptide of neuropeptide Y and tyrosine hydroxylase, all present in postganglionic sympathetic nerves, were not observed at any stage of antler growth. Nerves expressing immunoreactivity for any of the neural markers or peptides employed could not be found in cartilage, osteoid or bone. These results show that antlers are innervated mainly by sensory nerves and that nerves can attain the exceptionally high growth rates found in regenerating antler.

Animals↗
PubMed1992

Effect of retinoic acid on the resorptive activity of chick osteoclasts in vitro.

Mixed cell suspensions mechanically isolated from the long-bones of day 20 prehatch chicks were cultured for 24 h on bone and sperm whale dentine slices in the presence of 0, 10, 100, and 1000 nM retinoic acid (RA). Significant inhibitions in the numbers of discrete lacunae resorbed per dentine slice, and in the ratio of lacunae per tartrate-resistant acid phosphatase-positive multinuclear cell were observed with all concentrations of RA studied. Semi-automated, 3-dimensional analysis of 733 pits was performed on one series of experiments using a tandem scanning (confocal) microscope, interfaced to an image analyzing computer. The majority of lacunae were small and unilocular; the plan areas of 90% of control pits were below 500 microns 2. Small but statistically significant increases in lacunar areas, but not mean or maximum depths or volumes, were observed in the presence of 10 and 100 nM RA; however, these changes were much smaller than the magnitude of the decrease in pit numbers. Thus, the overall effect of RA in this system was inhibitory. Our findings contrast with the well known stimulatory action of retinoids on bone resorption both in vivo and in organ culture, but may parallel the inhibitory effects of prostaglandins observed in disaggregated osteoclast systems.

Animals↗
PubMed1991

Arg-Gly-Asp (RGD) peptides and the anti-vitronectin receptor antibody 23C6 inhibit dentine resorption and cell spreading by osteoclasts.

Studies with a range of monoclonal and polyclonal antisera to components of the human, rat, and chick vitronectin receptor, alpha V beta 3, and the VLA beta 1 chain show that chick and rat osteoclasts express similar integrin receptors to those described in man. Biochemical analysis with monoclonal antibody 23C6 confirmed the presence on chick osteoclasts of a vitronectin receptor heterodimer of similar size (110/95 kDa reduced) to that immunoprecipitated from human osteoclastoma giant cells. The synthetic peptide GRGDSP, corresponding to the cell adhesion sequence in fibronectin, but not GRGESP peptide, induced significant (P less than 0.005) osteoclast retraction in chick and rat osteoclasts at IC50s (+/- SEM) of 210.0 +/- 14.4 and 191.4 +/- 13.7 microM, respectively; monoclonal anti-vitronectin receptor alpha V beta 3 complex antibody, 23C6, produced similar changes in chick osteoclasts (IC50 = 1.45 +/- 0.22 microM). Antibody 23C6 inhibited the number of pits resorbed in dentine by chick osteoclasts over a concentration range of 4.4 to 88 micrograms/ml; a significant 76% reduction (P = 0.03) was observed at a final concentration of 88 micrograms/ml (6 microM). The effect of peptides upon dentine resorption was less dramatic. No consistent inhibition was seen using chick osteoclasts. Inhibitory effects on resorption by rat osteoclasts were, however, observed; significant reduction in resorption occurred with both GRGDSP (78%; P less than 0.01) and GRGESP (67%; P = 0.02) peptides at 400 microM peptide concentration. These data demonstrate that osteoclast function can be disrupted by low concentrations of the anti-vitronectin receptor antibody, 23C6. The inhibitory effects of the peptides used in this study produced effects on dentine resorption which were generally weaker and variable, although osteoclast cell adhesion was consistently inhibited in an Arg-Gly-Asp (RGD)-dependent manner. We conclude that the vitronectin receptor may play an important role in effecting resorption of mineralized tissues by osteoclasts.

Amino Acid Sequence↗
PubMed1989

Estrogen receptors and human bone cells: immunocytochemical studies.

In this immunocytochemical study we have probed a number of human bone cell types and bone preparations for the presence of the estrogen receptor (ER) with two distinct monoclonal antibodies. Using a well-validated antibody (H222) that recognizes human ER and standard peroxidase-antiperoxidase methodology, we were unable to demonstrate nuclear staining for ER in cultured primary or transformed human bone-derived cells or in fetal bone sections. Attempts to visualize ER in osteosarcoma cell lines (TE85C and HTB96) using a silver enhancement procedure were also unsuccessful. Additionally, we failed to detect immunocytochemical staining for the progesterone receptor (using monoclonal antibody mPR1) in control or estrogen-treated human bone cell cultures. Estrogen and progesterone receptor staining was readily detectable in MCF7 human breast cancer cells. In contrast, with a monoclonal antibody that recognizes a 29 kDa cytoplasmic component (p29) closely related to human ER, we observed specific staining in all the osteoblastlike cells studied. Cytoplasmic staining for this p29 antigen was most intense in primary cultures of human bone-derived cells. It is possible that the relatively abundant but as yet undefined p29 antigen may act as a sensitive marker for the presence of ER in cells at levels below the detection limit of the anti-ER monoclonal antibody. If so, our results are consistent with the presence of ER in osteoblastlike cells at very low concentrations.

Adult↗
PubMed1987

Biological activity of chicken calcitonin: effects on neonatal rat and embryonic chick osteoclasts.

Chicken calcitonin (cCT) has recently been synthesized according to nucleotide sequence data. We have investigated the in vitro effects of this hormone on the activity of disaggregated, neonatal rat and embryonic chick osteoclasts. While synthetic cCT inhibited bone resorption by neonatal rat osteoclasts at concentrations as low as 0.1 pg/ml, it failed to produce a significant reduction in bone resorption by embryonic chick osteoclasts, even at concentrations as high as 1 microgram/ml. Similarly, cCT at 1 pg/ml reproducibly produced the rapid and dramatic inhibition of rat osteoclast motility characteristic of other calcitonins, as judged by time-lapse video recording, but did not impair the motile behavior of chick osteoclasts at concentrations up to a million-fold higher. Previous studies showing that embryonic chick osteoclasts were unresponsive to synthetic salmon calcitonin left open the possibility that the native hormone was required to produce an inhibitory effect. We conclude that the osteoclast is not a target cell for calcitonin in the embryonic chick; further studies will be required to define a role, if any, for this potent but enigmatic hormone in birds.

Animals↗
PubMed1987

A comparative study of disaggregated chick and rat osteoclasts in vitro: effects of calcitonin and prostaglandins.

Disaggregated chick osteoclasts sedimented onto bovine cortical bone slices excavate deep and sharply defined resorption lacunae that stain intensely with toluidine blue. We have used this observation to develop a simple light microscopic method for quantifying the bone resorptive activity of chick osteoclasts in vitro. Using this technique, we have found that disaggregated chick osteoclasts are strikingly less sensitive than rat osteoclasts to salmon calcitonin (sCT). Bone resorption by rat osteoclasts was completely abolished by synthetic sCT at a concentration of 10 pg/ml. In contrast, sCT at concentrations up to 100 micrograms/ml did not significantly inhibit bone resorption by chick osteoclasts. Bone resorption by chick osteoclasts could, however, be inhibited by prostaglandin E2 at concentrations of 10(-6) M or more. In time-lapse video experiments, the motility of rat osteoclasts was rapidly inhibited by sCT (5-50 pg/ml), prostaglandins I2 and E2 (less than or equal to 10(-4) M), and Bu2cAMP (2.5 X 10(-4) M), whereas chick osteoclasts failed to show such a response. These findings suggest that CT does not play an important role in the regulation of osteoclastic activity in the chick and may explain why injection of the hormone has generally not been found to evoke an acute hypocalcemic response in birds. This fundamental difference in response to CT may limit the utility of chick osteoclast systems as models of mammalian bone resorption.

Animals↗
PubMed1986

Effect of pH on bone resorption by rat osteoclasts in vitro.

Acidosis has been implicated in the pathogenesis of various metabolic bone diseases. Experimental acid ingestion is known to stimulate the release of bone mineral in humans and increase osteoclastic resorption surfaces in animals, but the mechanism has remained unclear. We have investigated the effect of medium pH on osteoclastic activity in vitro using a new method that allows direct measurement of bone resorption. Osteoclasts were disaggregated from neonatal rat long bones and allowed to settle onto thin slices of bovine cortical bone. After 24-h incubation in medium 199, adjusted to the required pH, bone slices were fixed, and the number and surface area of resorption pits per slice were determined. Reduction in medium pH from 7.4 to 6.8 resulted in a 14-fold increase in the mean area resorbed per bone slice (P less than 0.01). Five- and 9-fold increases were seen at pH 7.2 and 7.0, respectively. Incubation of bone cells with human PTH-(1-34) (100 ng/ml) did not significantly enhance resorption at either pH 7.4 or 6.4, whereas human calcitonin (50 pg/ml) produced near-total inhibition. Mean osteoclast and mononuclear cell numbers were unaltered by all treatments. This is the first direct demonstration that low pH stimulates cell-mediated bone resorption, an observation that may have considerable physiological and pathophysiological significance.

Acidosis↗